This invention relates to a cDNA encoding a tapasin-like protein and to the use of the cDNA, protein, and an antibody binding the protein in the diagnosis, prognosis, treatment and evaluation of therapies for cancers, particularly of the prostate, bladder and lung, and immune response.
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of biochemical and physiological mechanisms and metabolic pathways. Despite different evolutionary pressures, proteins that regulate the cell cycle in yeast, nematode, fly, rat, and man have common chemical or structural features and modulate the same general cellular activity. Comparisons of human gene sequences with those from other organisms where the structure and/or function may be known allow researchers to draw analogies and to develop model systems for testing hypotheses. These model systems are of great importance in developing and testing diagnostic and therapeutic agents for human conditions, diseases and disorders.
Tapasin is a 48-kDa transmembrane glycoprotein. It is found in the endoplasmic reticulum (ER) and displays a cytoplasmic retention signal. Tapasin is a member of the immunoglobulin (Ig) superfamily and is encoded by an major histocompatibility (MHC)-linked gene. The protein plays a critical functional role in MHC class I-restricted antigen processing. Tapasin mediates the interaction between the transporter associated with antigen processing (TAP) and newly synthesized MHC class I molecules by forming complexes with other chaperones such as calnexin and calreticulin. Up to four MHC class I-tapasin complexes bind and present molecules to each TAP molecule. (See Pamer and Cresswell (1998) Annu Rev Immunol 16:323-58; Ortmann et al. (1997) Science 277:1306-9.)
Tapasin is essential for human lymphocyte (HLA) Al, B8, and B4402 antigen presentation. Although tapasin is required for HLA-A2 molecules to bind TAP, its absence affects the overall efficiency of the process of loading HLA-A2 with optimal, stabilizing peptides. With its Ig_MHC binding signature, (Y)xCx(V)xH, tapasin is a necessary cofactor in a multicomponent ""peptide loading complex"" where lack of binding results in proteasome-mediated degradation (Lewis et al. (1998) Eur J Immunol 28:3214-20). After analysis of mutant molecules which fail to bind tapasin or TAP, Suh et al. (1999; J Immunol 162:1530-40) also suggested a peptide-editing function for tapasin/TAP in addition to a role in enhancing peptide loading.
Correct antigen presentation to T lymphocytes is important in the infectious disease process. In a study of the mutant MHC class I molecule T134K (in which Thr134 was changed to Lys), Lewis and Elliott (1998; Curr Biol 8:717-20) reported that the point mutation disrupted, directly or indirectly, the interaction between MHC class I molecules and calreticulin. T134K molecules were transported out of the ER as ""empty"" MHC class I complexes rather than being retained and degraded and neither bound TAP nor presented viral antigens to T cells.
The discovery of a cDNA encoding a tapasin-like protein provides compositions which are useful in the diagnosis, prognosis, treatment and evaluation of therapies for cancers, particularly of the prostate, bladder and lung, and immune response.
The present invention is based on the discovery of a cDNA encoding a tapasin-like protein (TLP). The cDNA which encodes the protein, and an antibody which specifically binds the protein are useful in the diagnosis, prognosis, treatment and evaluation of therapies for cancers, particularly of the prostate, bladder and lung, and immune response.
The invention provides an isolated cDNA comprising a nucleic acid sequence of SEQ ID NO:1 which encodes a protein having an amino acid sequence of SEQ ID NO:2. The invention also provides isolated cDNA fragments comprising SEQ ID NOs:3-9 which have about 100% sequence identity with SEQ ID NO:1. The invention additionally provides isolated cDNAs comprising SEQ ID NOs:10-13 which have from about 75% to about 90% nucleotide identity with SEQ ID NO:1. The invention further provides complements of SEQ ID NOs:1 and 3-13.
The invention provides compositions comprising the cDNAs or complements thereof and a labeling moiety or pharmaceutical carrier which may be used in methods of the invention, on a substrate, as probes or therapeutics. The invention also provides a vector containing the cDNA, a host cell containing the vector, and a method for using the cDNA to make tapasin-like protein. The invention additionally provides a transgenic cell line or organism comprising the vector containing a cDNA selected from SEQ ID NO:1 and 3-13. The invention further provides for using a cDNA or the complement thereof in methods of detection, screening, and purification. In one embodiment, the cDNA is a single-stranded RNA or DNA molecule, a peptide nucleic acid, a branched nucleic acid, and the like.
The invention provides a method for using a cDNA to detect differential expression of a nucleic acid in a sample comprising hybridizing a cDNA to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with at least one standard, wherein the comparison indicates differential expression of the cDNA in the sample. In one aspect, the method further comprises amplifying the nucleic acids of the sample prior to hybridization. In a second aspect, the method is used to diagnose a cancer.
The invention additionally provides a method for using a cDNA or composition of the invention to screen a plurality of molecules or compounds to identify or purify at least one ligand which specifically binds the cDNA or composition, the method comprising combining the cDNA or composition with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the cDNA or composition, thereby identifying or purifying a ligand which binds the cDNA or composition. In one embodiment, the molecules or compounds are selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, transcription factors, repressors, and regulatory molecules.
The invention provides a purified protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:2, a variant of SEQ ID NO:2, an antigenic epitope of SEQ ID NO:2, and a biologically active portion of SEQ ID NO:2. The invention also provides a composition comprising purified protein and a labeling moiety or a pharmaceutical carrier. The invention further provides a method of using a protein to treat a subject with cancer comprising administering to a patient in need of such treatment a composition containing purified protein and a pharmaceutical carrier. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify or purify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying or purifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs. In a second aspect, the ligand is used to treat a subject with a cancer.
The invention provides a method of using a protein having the amino acid sequence of SEQ ID NO:2 to screen a plurality of antibodies to identify and purify an antibody which specifically binds the protein comprising contacting isolated antibodies with the protein under conditions to form an antibody:protein complex, and dissociating the antibody from the protein, thereby obtaining purified antibody which specifically binds the protein.
The invention also provides a method of using a protein having the amino acid sequence of SEQ ID NO:2 to prepare and purify polyclonal and monoclonal antibodies which specifically bind the protein. The method for using the protein to prepare a polyclonal antibody comprises immunizing a animal with protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified polyclonal antibodies. The method for using the protein to prepare monoclonal antibodies comprises immunizing an animal with a protein under conditions to elicit an antibody response, isolating antibody producing cells from the animal, fusing the antibody producing cells with immortalized cells in culture to form monoclonal antibody producing hybridoma cells, culturing the hybridoma cells, and isolating from culture monoclonal antibodies which specifically bind the protein.
The invention provides purified polyclonal and monoclonal antibodies which bind specifically to a tapasin-like protein. The invention also provides a method for using an antibody to detect expression of a protein in a sample, the method comprising combining the antibody with a sample under conditions which allow the formation of antibody:protein complexes; and detecting complex formation, wherein complex formation indicates expression of the protein in the sample. In one aspect, the amount of complex formation when compared to standards is diagnostic of cancer.
The invention provides a method for inserting a heterologous marker gene into the genomic DNA of a mammal to disrupt the expression of an endogenous polynucleotide. The invention also provides a method for using a cDNA to produce a model system, the method comprising constructing a vector containing a DNA selected from SEQ ID NOs:1 and 3-13, transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem cell, microinjecting the transformed embryonic stem cell into a blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, model system.